Theor Appl Genet DOI 10.1007/s00122-014-2361-4

Original Paper

Mapping resistance genes for Oculimacula acuformis in longissima

Hongyan Sheng · Deven R. See · Timothy D. Murray

Received: 27 February 2014 / Accepted: 13 July 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract chromosomal locations as some for resistance to O. yallun- Key message This study identified three QTL confer- dae, except that a QTL for resistance to O. yallundae was ring resistance to Oculimacula acuformis in Aegilops found in chromosome 7Sl but not for O. acuformis. Thus, longissima and their associated markers, which can be it appears that some genes at the same locus in Ae. long- useful in marker-assisted selection breeding for eyespot issima may control resistance to both eyespot pathogens. resistance. QTL effective against both pathogens will be most useful Abstract Oculimacula acuformis is one of two for breeding programs and have potential to improve the of soilborne fungi that cause eyespot of wheat, the other effectiveness and genetic diversity of eyespot resistance. being Oculimacula yallundae. Both pathogens can coex- ist in the same field and produce elliptical lesions on stem bases of wheat that are indistinguishable. Pch1 and Pch2 Introduction are the only two eyespot resistance genes readily avail- able to wheat breeders, but neither provides complete Eyespot, a stem base disease of wheat, is caused by Ocu- control. A new source of eyespot resistance was identified limacula acuformis Crous & W. Gams (syn: Tapesia acu- from Aegilops longissima (2n 14, SlSl), a wild relative formis, Wallwork & Spooner) and O. yallundae Crous & = of wheat. Three QTL for resistance to O. acuformis were W. Gams (syn: T. yallundae) (Crous et al. 2003). These mapped in chromosomes 1Sl, 3Sl, and 5Sl using a recom- fungi often coexist in the same field and cause eye-shaped binant inbred line population developed from the cross Ae. elliptical lesions at the stem bases that are indistinguish- longissima accessions PI 542196 (R) PI 330486 (S). able. Eyespot occurs in many cool and wet wheat growing × The three QTL explained 66 % of phenotypic variation areas in the world. In the USA, eyespot is a chronic and by β-glucuronidase score (GUS) and 84 % by visual rat- economically important disease of winter wheat in the ing. These QTL had LOD values of 10.6, 8.8, and 6.0 for Pacific Northwest (PNW) region including Idaho, Oregon GUS score, and 16.0, 10.0, and 13.0 for visual rating. QTL and Washington. Severe eyespot results in stem breakage associated with resistance to O. acuformis have similar and multi-directional lodging in the field (Maloy and Inglis 1993). Up to 50 % yield reduction has been documented in severely affected fields (Murray 2010). Communicated by Frank Ordon. Although eyespot was controlled with fungicides for many years, limiting input costs, environmental concerns, * H. Sheng · D. R. See · T. D. Murray ( ) and fungicide resistance resulted in the need for planting Department of Pathology, Washington State University, Pullman, WA 99164‑6430, USA resistant cultivars. Resistance to eyespot has been investi- e-mail: [email protected] gated since the pathogen was first described (Sprague and Fellowes 1934). Discovery of the wheat relative Aegilops D. R. See ventricosa. Tausch (2n 28, DDMvMv) (syn. Triticum US Department of Agriculture, Agricultural Research Service = (USDA‑ARS), Wheat Genetics, Quality Physiology and Disease ventricosum (Tausch) Ces.) by Sprague (1936) led to the Research Unit, Pullman, WA 99164‑6430, USA introgression of eyespot resistance gene Pch1 into common

1 3 Theor Appl Genet wheat (Kimber 1967; Doussinault et al. 1983). Pch1 is the Aegilops longissima Schweinf. & Muschl. (2n 2x 14, = = most effective resistance gene to eyespot to date. It is a sin- SlSl), a diploid species in the section Sitopsis of Aegilops L. gle dominant gene mapped to the distal end of the long arm (Van Slageren 1994), is a distant relative of wheat and poten- of chromosome 7D (Worland et al. 1988). Through breed- tial donor of genes for wheat cultivar improvement, including ing line VPM-1 (Ventricosa Persicum Marne), Pch1 disease resistance (Friebe et al. 1993). In previous studies, × × has been incorporated into several commercial wheat cul- Ae. longissima was identified as containing a new source of tivars for use in the US PNW. Madsen, the first eyespot eyespot resistance (Sheng and Murray 2013). Multiple genes resistant line in the US PNW, was released in 1988 by the in the Sl genome were found to be responsible for genetic USDA-ARS and Washington State University winter wheat control of eyespot resistance and some of them reacted dif- breeding program at Pullman, WA (Allan et al. 1989). It has ferently from O. yallundae and O. acuformis (Sheng and been widely grown in the PNW for more than two decades Murray 2013). A genetic linkage map composed of 169 (Murray 2010). Lind (1999) found that Pch1 resistance was wheat microsatellite markers (SSR markers) was constructed more effective in early plant growth stages than at the adult with the Sl genome with a recombinant inbred line popula- stage. Burt et al. (2010) found that Pch1 conferred resist- tion of Ae. longissima (Sheng et al. 2012). Furthermore, four ance to both O. acuformis and O. yallundae at the seedling QTL conferring resistance to O. yallundae were mapped in stage. chromosome 1Sl, 3Sl, 5Sl, and 7Sl (Sheng et al. 2012). The French variety Cappelle Desprez containing Pch2 is objective of this study was to determine the genetic control the only known source of eyespot resistance from hexa- of resistance to O. acuformis in the Sl genome of Ae. longis- ploid wheat (Hollins et al. 1988). Law et al. (1976) found sima by QTL mapping using wheat SSR markers. This work evidence that eyespot resistance in Cappelle Desprez is will contribute to the long-term goal of transferring new eye- located on chromosomes 1A, 2B, 5D, and 7A, with a major spot resistance genes to wheat. component on 7A. Pch2 was later mapped to the distal por- tion of 7AL with RFLP markers (de la Peña et al. 1997). Recently, a major QTL associated with eyespot resistance Materials and methods was mapped on chromosome 5AL of Cappelle Desprez (Burt et al. 2011). Although Pch2 is less effective than Mapping population Pch1 (Johnson 1992), Cappelle Desprez has been grown extensively and its resistance has been transferred to many Aegilops longissima accessions PI 542196 and PI 330486 other wheat varieties in Europe since the 1950s (Hollins provided by the USDA National Small Grains Collection et al. 1988). Burt et al. (2010) found that Pch2 was signifi- (NSGC) were identified as resistant and susceptible to cantly more effective against O. acuformis than O. yallun- both O. yallundae and O. acuformis, respectively (Sheng dae at the seedling stage and that the QTL on chromosome and Murray 2013), and were selected as the resistant 5A conferred resistance to both pathogens at the seedling and susceptible parents for development of a recombi- and adult stages (Burt et al. 2011). nant inbred line (RIL) population (Sheng et al. 2012).

Although Pch1 and Pch2, along with the QTL on 5AL The population was developed to F8 through single-seed of Cappelle Desprez, have been used to effectively pro- descent. tect wheat from eyespot disease, none of them provides complete resistance to eyespot when used alone. Addi- Phenotypic evaluation tional resistance genes are desired for incorporation into adapted wheat cultivars to improve the effectiveness and One-hundred and seventy-eight F RIL of PI 542196 (R) 8 × diversity of resistance genes. Wild species of wheat have PI 330486 (S) were tested for resistance to O. acuformis received considerable attention as sources of eyespot resist- in two growth chamber (Controlled Environments Lim- ance (Jones et al. 1995). A single dominant gene, Pch3 or ited, Winnipeg, Manitoba, Canada) experiments. The PchDv, was mapped to chromosome 4VL of Dasypyrum parent lines and the winter wheat cultivar Madsen were villosum because of its eyespot resistance (Yildirim et al. included in the experiments as controls. Each experiment 1998), but the gene has not been used commercially. Uslu was a randomized complete block (RCB) design with two et al. (1998) found multiple resistance genes in D. villo- subsamples and three blocks. Thus, a total of 12 per sum chromosomes 4V and 5V to O. yallundae and O. acu- line were evaluated for phenotypic variation. The planting formis, respectively; the first report showing differential method and growth chamber conditions were the same as resistance to these two pathogens. Burt et al. (2010) also for the phenotypic evaluation of F5 RIL to O. yallundae demonstrated that Triticum monococcum accessions had (Sheng et al. 2012). significantly different resistance reactions to O. yallundae Disease evaluation was conducted after inoculating and O. acuformis. seedlings with β-glucuronidase (GUS) transformed O.

1 3 Theor Appl Genet acuformis isolates tph98-2-34A, tph98-2-34D, tph98-2- WinQTLCart V2.5 (Wang et al. 2010). Composite Interval 34E, and tph98-1-54aa (de la Peña and Murray 1994). Mapping was conducted to identify the chromosome loca- Each two-leaf stage seedling was inoculated twice with tions of major QTL associated with resistance to O. acu- 250 µl slurry of conidia containing 1.2 105 conidia per formis (Sheng et al. 2012). × ml (Sheng et al. 2012). Visual disease ratings were performed at growth stage 23–25 (Zadoks et al. 1974), about 8 weeks after inocula- Results tion. All tillers (2–4) of each plant were cut into a 3 cm- long section above the soil line. After washing in water Phenotypic evaluation to remove soil, stems were evaluated visually for disease severity on a 0–4 scale, where 0 healthy to 4 nearly Based on the F ratio test, the error variances of GUS scores = = dead (Sheng et al. 2012). GUS activity in stems, a surro- and visual ratings were not significantly different at 95 % gate measurement of fungal growth, was determined with a significance level between the two experiments. Therefore, fluorescent GUS assay using a methylumbelliferone (MU) homogeneity of variance was accepted for both GUS scores substrate. Fluorescence was measured in a SpectraMax and visual ratings, and data from the two experiments were M2 microplate reader (Molecular Devices Co., Sunnyvale, combined for further analysis. The mean GUS scores were CA). Each sample was ground with 2.5 ml GUS extrac- normally distributed (KS test, P > 0.15) and ranged from tion buffer (Sheng et al. 2012). Fluorescence intensity was 0.8 to 1.8 (Fig. 1). Mean visual ratings were also normally expressed as the log10 transformed ratio [log10(x/resistant distributed and ranged from 0.8 to 3.7 (Fig. 1). control) 1] (Sheng et al. 2012) of an individual accession β-Glucuronidase score (GUS) scores were significantly + (x) compared to the resistant control (Madsen). correlated (r 0.7025, P < 0.0001) with visual ratings in = the F8 population. The resistant parent (PI 542196) had sig- Statistical analysis nificantly (P < 0.0001) lower GUS scores and visual ratings

SAS Version 9.2 (SAS Institute Inc., Cary, NC) was used for statistical analysis. Homogeneity of variance of the two experiments was tested and if the hypothesis of homoge- neity was accepted, data from the two experiments were combined for further analysis (Gomez and Gomez 1984). PROC GLM was used for analysis of variance (ANOVA) and standard deviation of two dependent variables (GUS score and visual rating). Variance components were gener- ated by a random model with lines and experiments as ran- dom effects in ANOVA. The Kolmogorov-Smirnov (KS) test was used to test normality of the phenotype distribu- tion. Dunnett’s t test was used for multiple comparisons of the least squares mean (lsmean) of each RIL compared with the resistant parent (PI 542196). The genotypes based on presence of specific QTL were compared by Tukey’s t test with least squares means (lsmean). The correlation between visual ratings and GUS scores was evaluated by Pearson correlation coefficients. Broad-sense heritability (H2) was calculated as the ratio of the genetic variance [Var(G)] to the phenotypic variance [Var(P)] (Sheng et al. 2012).

Linkage mapping and QTL analysis

In the previous study (Sheng et al. 2012), a genetic linkage map of Ae. longissima was established based on the geno- Fig. 1 Distribution of GUS scores and visual ratings in F population typic data of 178 F5 RIL of (PI 542196 PI 330486) with 8 × (178 lines, 12 plants/line) of the cross PI 542196 (R) PI 330486 169 wheat microsatellite (SSR) markers. Based on seven (S) inoculated with Oculimacula acuformis. The null hypothesis× of linkage groups of that map and phenotypic data of same normality was accepted based on the Kolmogorov–Smirnov test with RIL advanced to the F8, QTL analysis was performed with values of 0.0475 and 0.0559 (P > 0.15)

1 3 Theor Appl Genet

Table 1 Variance components of GUS scores and visual ratings and gwm642, cfd48, barc128, gwm32, and cfd83) covering 2 broad-sense heritability (H ) for F8 recombinant inbred lines (RIL) 30.3 cm. All eight markers were significantly associ- derived from the cross PI 542196 (R) PI 330486 (S) and inoculated l with Oculimacula acuformis × ated with Q.Pch-oa.wsu-1S (P < 0.0001). Markers cfd6, gdm67, gwm642, cfd48, barc128, and gwm32, which Source of df GUS score Visual rating were clustered in an 8.7 cm interval on chromosome 1Sl, variation Mean F value Mean F value were most closely linked to Q.Pch-oa.wsu-1Sl. An addi- square square tive effect (0.13 for GUS score and 0.43 for visual rat- RILa 177 0.49 4.75** 4.19 3.66** ing) was contributed from the resistant parent PI 542196. Block (expt.) 4 7.3 70.8** 27.32 23.85** Markers gwm642 and cfd48 are located on the long arms Experiment 1 57.66 7.9 35.89 1.31 of wheat chromosome 1D and 1B (GrainGenes, http:// Expt. RIL 177 0.198 1.92** 2.57 2.24** wheat.pw.usda.gov/cgi-bin/graingenes), respectively, × l Error 1,730 0.103 1.146 indicating that Q.Pch-oa.wsu-1S is on the long arm of chromosome 1Sl. At markers gdm67, gwm642 and H2 (based on 69.4 % 56.6 % line means) cfd48, the mean GUS score and visual rating of lines with the resistant allele (PI 542196) were significantly ** P < 0.0001 (P < 0.0001) less than lines with the susceptible allele a 178 lines; 12 plants/line were included in two experiments (PI 330486) (Fig. 3). Q.Pch-oa.wsu-3Sl, located on chromosome 3Sl, had than the susceptible parent (PI 330486). PI 542196 had LOD values of 8.8 and 10.0 for GUS score and visual rat- mean GUS score (1.1) and mean visual rating (1.1), which ing and explained 27 and 30 % of the phenotypic variation, were not significantly (P > 0.05) different from those of respectively (Fig. 2b). Q.Pch-oa.wsu-3Sl was associated Madsen (1.0 and 1.2, respectively). The mean GUS score with both GUS score and visual rating in a 40.6 cm inter- (1.6) and visual rating (3.5) of PI 330486 were both sig- val between markers cfd79 and barc71. Marker cfd9 was in nificantly (P < 0.0001) greater than the mean values for an interval about 6 cm from the QTL peak. All three mark- Madsen. ers (cfd79, cfd9, and barc71) had significant (P < 0.0001) The variance components of GUS scores and visual rat- effects on both GUS score and visual rating. The additive ings of F8 RIL were determined by analysis of variance effects from the resistant parent PI 542196 were 0.11 and (ANOVA). There were significantly (P < 0.0001) different 0.34 for GUS score and visual rating, respectively. Q.Pch- l reactions to O. acuformis among the 178 F8 RIL for both oa.wsu-3S probably is located on the short arm of chromo- GUS score and visual rating (Table 1). The environment some 3Sl since markers gwm314, barc147, and cfd79 are effect (block within experiment) and genotype by envi- either on 3BS or 3DS of wheat chromosomes according to ronment interaction (Expt. RIL) were also significant GrainGenes (http://wheat.pw.usda.gov/cgi-bin/graingenes). × (P < 0.0001) for both. However, experiments were not sig- The mean GUS score and visual rating were signifi- nificantly different for either GUS score (P 0.1597) or cantly (P < 0.0029 and P < 0.0044, respectively) different = visual rating (P 0.3156). The F population demonstrated between lines with opposite alleles of markers cfd79, cfd9, = 8 69.4 % broad-sense heritability (H2) based on line means and barc71 (Fig. 3). for the reaction to O. acuformis by GUS score and 56.6 % Q.Pch-oa.wsu-5Sl had LOD values of 6.0 and 13.0 for by visual rating (Table 1). GUS score and visual rating, and explained 12 and 23 % of the phenotypic variation, respectively (Fig. 2c). Q.Pch- QTL analysis oa.wsu-5Sl was between markers wmc415 and barc319 in a 24.9 cm interval. Six markers (wmc415, cfd12, gwm408, Three QTL conferring resistance to O. acuformis were gwm271, wmc160, and barc319) fell in this interval and identified on chromosomes 1Sl, 3Sl, and 5Sl (Fig. 2a–c). All all were significantly (P < 0.0001) associated with Q.Pch- of them were detected using both GUS scores and visual oa.wsu-5Sl by GUS score and visual rating. Markers cfd12, ratings and were contributed by the resistant parent, PI gwm408 and gwm271 are in a 6.3-cm interval and the most 542196. These QTL were designated as Q.Pch-oa.wsu-1Sl, closely linked. Additive effects were 0.06 for GUS score Q.Pch-oa.wsu-3Sl, and Q.Pch-oa.wsu-5Sl. and 0.29 for visual rating, which came from the resistant The QTL detected on chromosome 1Sl, Q.Pch- parent PI 542196. Q.Pch-oa.wsu-5Sl probably is on the long oa.wsu-1Sl, had LOD values of 10.6 and 16.0 for GUS arm of chromosome 5Sl since the five markers associated score and visual rating and explained 27 and 31 % of with this QTL are located on wheat chromosomes 5AL, the phenotypic variation of GUS score and visual rating, 5BL or 5DL (http://wheat.pw.usda.gov/cgi-bin/graingenes). respectively (Fig. 2a). SSR markers cfd6 and gdm132 At the closest linked markers cfd12, gwm408, and gwm271, flanked this QTL, spanning six other markers (gdm67, the mean GUS score and visual rating were significantly

1 3 Theor Appl Genet

PI 330486 PI 542196 P = 0.05 A 1.6

1.5

1.4 A 1.3 AA A A A AA A GUS score 1.2 Xg Xg Xc B B B B fd dm67 wm64 1.1 B B B B 48 B

2 1.0

2.9

2.7

2.5 A 2.3 A A rating AA A B 2.1 A AA sual

Vi 1.9 BB 1.7 B B B B B B B 1.5

Xc Xc Xbarc7 1 Fig. 3 GUS scores and visual ratings for RIL of PI 542196 (R) PI fd fd

79 9 330486 (S) with different parental alleles at the markers close to ×each QTL. gdm67, gwm642, and cfd48 are close to Q.Pch-oa.wsu-1Sl; cfd79, cfd9, and barc71 are close to Q.Pch-oa.wsu-3Sl; and cfd12, gwm408, and gwm271 are close to Q.Pch-oa.wsu-5Sl. The dark and light bars represent the mean GUS scores or visual ratings of RIL with the susceptible and resistant allele at the marker closet to each QTL, respectively. They were all significantly different (P < 0.05) between susceptible and resistant allele at the closest markers which C were indicated by different letters on the bars. Error bars show stand- ard errors

(P < 0.0045) less for the resistant parental alleles than the susceptible alleles (Fig. 3). Eight genotypes, defined by combinations of the resistant parental allele at the closest marker to each QTL, were pro- Xcfd12 Xg Xg duced from 178 RIL with 13–33 lines each. Markers cfd48, wm408 wm271 cfd9, and gwm271 are the closest markers to Q.Pch-oa.wsu- 1Sl, Q.Pch-oa.wsu-3Sl, and Q.Pch-oa.wsu-5Sl, respectively. The LSmeans for GUS score and visual rating for each genotype were calculated (Fig. 4) and were significantly (P < 0.0001) different among the eight genotypes. Lines with no QTL had the greatest GUS score (1.5) and visual rating (3.0), and the lines with all QTL had the lowest GUS Fig. 2 QTL for resistance to Oculimacula acuformis identified on score (1.1) and visual rating (1.6). All the QTL genotypes Aegilops longissima chromosomes 1Sl, 3Sl, and 5Sl by GUS score had significantly (P < 0.0001) lower disease severity than and visual rating with composite interval mapping. a Q.Pch-oa.wsu- the genotype with no QTL. Although the GUS scores and l l l l 1S on chromosome 1S ; b Q.Pch-oa.wsu-3S on chromosome 3S ; and visual ratings for genotypes with one QTL were not signifi- c Q.Pch-oa.wsu-5Sl on chromosome 5Sl. Dashed lines represent the map locations of Q.Pch.wsu-1Sl, Q.Pch.wsu-3Sl, and Q.Pch.wsu-5Sl cantly different (P > 0.1975 and P > 0.1051, respectively) to O. yallundae, respectively (Sheng et al. 2012) from each other, they all had significantly (P < 0.0266) 1 3 Theor Appl Genet

1.6 5Sl that explained 66 and 84 % of the phenotypic varia- P = 0.05 tion in GUS scores and visual ratings, respectively. This 1.5 result demonstrates that the resistance to O. acuformis in

1.4 Ae. longissima accession PI 542196 is controlled by multi- e ple QTL. It is also consistent with the finding that multiple 1.3 QTL confer the resistance to O. yallundae in Ae. longis- sima (Sheng et al. 2012). The genetic control of eyespot 1.2 GUS scor * resistance in Ae. longissima is polygenic and the genes * are quantitative. Eyespot resistance in Cappelle Desprez is 1.1 * * * also polygenic (Law et al. 1976; Jahier et al. 1979) but the 1.0 quantitative character was found only on chromosome 5AL

3.5 (Burt et al. 2011). Environment and genotype by environment interactions

3.0 were significant in the phenotypic tests in this study. A significant block effect occurred because different growth g 2.5 chambers were used. Other factors contributing to the

ratin block effect were variation in humidity within chambers 2.0 * * and the disease evaluation period, which took about 1 week sual *

Vi to complete; experiments were evaluated by block, but con- * 1.5 ** tinued disease development during this time would result in * block variation. l 1.0 Q.Pch-oa.wsu-1S to O. acuformis was detected on chro- No QTL 1S 3S 5S 1S + 3S 1S + 5S 3S + 5S All QTL mosome 1Sl and eight SSR markers were significantly associated with it. Five markers (gdm67, gwm642, cfd48, Fig. 4 Mean GUS scores and visual ratings of RIL within each geno- barc128, and gwm32) are co-dominant markers and four type. Resistance of eight genotypes based on three QTL detected of them (gwm642, cfd48, barc128, and cfd83) were also in F8 RIL populations of PI 542196 (R) PI 330486 (S) to Oculi- macula acuformis. Each genotype includes× 13–33 lines and each mapped on wheat homoeologous chromosomes 1B or 1D line includes 12 plants. 1S, 3S, and 5S represent Q.Pch-oa.wsu- (Somers et al. 2004). We found that the physical arrange- 1Sl, Q.Pch-oa.wsu-3Sl, Q.Pch-oa.wsu-5Sl, and Q.Pch.wsu-7Sl, ments of markers gwm642, cfd48, and barc128 in Ae. long- respectively. Bars with an asterisk are genotypes with significantly issima and wheat were collinear. Q.Pch.wsu-1Sl associated (P < 0.05) lower GUS scores or visual ratings than ‘no QTL’. Light bars are genotypes that were not significantly (P > 0.05) greater than with resistance to O. yallundae was mapped in a similar l l ‘all QTL’ for either GUS score or visual rating. Error bars show location on chromosome 1S as Q.Pch-oa.wsu-1S with a standard errors 13.5 cm overlap (Sheng et al. 2012) and shared seven mark- ers (cfd6, gdm67, gwm642, cfd48, barc128, gwm32, and cfd83). Markers cfd6, gdm67, gwm642, and cfd48 were lower visual ratings than the genotype with no QTL; how- most closely linked to both Q.Pch.wsu-1Sl and Q.Pch- ever, only Q.Pch-oa.wsu-5Sl had significantly (P 0.0024) oa.wsu-1Sl. It is possible that Q.Pch.wsu-1Sl and Q.Pch- = lower GUS score than the no QTL genotype. All single QTL oa.wsu-1Sl are linked to the same genes or different genes at genotypes had significantly (P < 0.0003) greater GUS score the same locus. The LOD values of Q.Pch-oa.wsu-1Sl were and visual rating than the genotype with all QTL. greater than those of Q.Pch.wsu-1Sl for GUS score and vis- Three genotypes with combinations of two QTL had sig- ual rating. Q.Pch-oa.wsu-1Sl also explained 16 % more phe- nificantly (P < 0.0035) lower GUS scores and visual ratings notypic variation than Q.Pch.wsu-1Sl for both GUS score than the genotype with no QTL and were not significantly and visual rating. Thus, the gene on chromosome 1Sl may different (P > 0.3106 and P > 0.5762, respectively) from be more effective to O. acuformis than to O. yallundae. each other. Only the combination of Q.Pch-oa.wsu-1Sl and The region containing both Q.Pch.wsu-1Sl and Q.Pch- Q.Pch-oa.wsu-3Sl was not significantly (P 0.0515) dif- oa.wsu-1Sl was independently mapped to the long arm of = ferent from the genotype of all QTL for GUS score (Fig. 4). Ae. longissima chromosome 1Sl (Sheng et al. 2012). Resist- ance to both O. yallundae and O. acuformis was also found on the long arm of Ae. longissima chromosome 1Sl by eval- Discussion uating Ae. longissima ditelosomic addition and substitution lines (Sheng and Murray 2013). The resistant lines will be Three QTL contributing resistance to O. acuformis were validated with the markers associated with the QTL identi- detected on Ae. longissima chromosomes 1Sl, 3Sl, and fied in PI 542196. Overall, it can be concluded that the long

1 3 Theor Appl Genet arm of Ae. longissima chromosome 1Sl contains genes that a similar location on chromosome 5Sl as Q.Pch-oa.wsu- confer eyespot resistance to both fungal pathogen species 5Sl with a 9.5 cm overlap (Sheng et al. 2012). These QTL and are quantitatively inherited. shared three markers (wmc415, cfd12, and gwm408), which Besides Ae. longissima, homoeologous chromosome were the most closely linked markers to both Q.Pch.wsu- group 1 was found to contain eyespot resistance in other 5Sl and Q.Pch-oa.wsu-5Sl. Thus, these two QTL may rep- resources. A minor effect to eyespot on chromosome 1A of resent one gene or two different genes at the same locus. Cappelle Desprez was reported (Law et al. 1976) but was Nine markers were associated with the region contain- not specifically for O. yallundae or O. acuformis. Later, ing Q.Pch.wsu-5Sl and Q.Pch-oa.wsu-5Sl on chromosome chromosome 1V from Dasypyrum villosum, a wild species 5Sl; all are located on wheat homoelogous group 5 chro- of wheat, was found to confer resistance to both O. yallun- mosomes in collinear order (Somers et al. 2004). Both dae and O. acuformis (Uslu et al. 1998). From our studies, Q.Pch.wsu-5Sl and Q.Pch-oa.wsu-5Sl were mapped on the the presence of resistance on homoeologous chromosome long arm of Ae. longissima chromosome 5Sl in two dif- group 1 was confirmed. ferent studies (Sheng et al. 2012) and to chromosome arm Q.Pch-oa.wsu-3Sl was significantly associated with 5SlL with Ae. longissima ditelosomic addition and substitu- three SSR markers on chromosome 3Sl, which are also tion lines (Sheng and Murray 2013). located in wheat homoeologous group 3 (Somers et al. Law et al. (1976) reported that chromosome 5D of 2004). Marker cfd79 along with two other markers at the Cappelle Desprez contributed to eyespot resistance, but it distal end of the short arm has co-linearity with wheat chro- was not characterized since it was less effective than the mosome 3D. Q.Pch.wsu-3Sl was detected 9 cm proximal to gene on chromosome 7AL. Muranty et al. (2002) identi- Q.Pch-oa.wsu-3Sl on chromosome 3Sl (Sheng et al. 2012). fied quantitative eyespot resistance on chromosome 5A in It is possible that these QTL represent two different genes Cappelle Desprez. Furthermore, Burt et al. (2011) mapped controlling resistance to the two Oculimacula species. The a major QTL conferring resistance to both O. yallun- LOD values of Q.Pch-oa.wsu-3Sl were greater than those dae and O. acuformis on chromosome 5AL in Cappelle of Q.Pch.wsu-3Sl for GUS score and visual rating and Desprez. They also found that the most closely linked SSR Q.Pch-oa.wsu-3Sl explained 13 and 21 % more phenotypic marker was gwm639. In our studies, gwm639 was one of variation than Q.Pch.wsu-3Sl for GUS score and visual rat- the closest markers associated with Q.Pch.wsu-5Sl but ing, respectively. These results suggest that Q.Pch-oa.wsu- not Q.Pch-oa.wsu-5Sl on chromosome 5SlL (Sheng et al. 3Sl may be associated with a more effective gene than the 2012). However, the flanking marker of Q.Pch-oa.wsu-5Sl, gene conditioning resistance to O. yallundae. wmc415, was mapped at the distal side of gwm639 about Both Q.Pch-oa.wsu-3Sl and Q.Pch.wsu-3Sl were mapped 6.7 cm away. The other flanking marker of Q.Pch-oa.wsu- to the short arm of Ae. longissima chromosome 3Sl (Sheng 5Sl, barc319, which was 31.6 cm away from gwm639 on et al. 2012). Resistance to O. acuformis was also identified 5SlL, was 27 cm on the distal side of chromosome 5AL in on the short arm of Ae. longissima chromosome 3Sl when Cappelle Desprez (Burt et al. 2011). Marker gwm639 was Ae. longissima ditelosomic addition and substitution lines linked to a QTL for Fusarium head blight on wheat chro- were tested (Sheng and Murray 2013). However, those lines mosomes 5A and 5B in two different studies, respectively did not reveal any evidence of resistance to O. yallundae on (Gervais et al. 2003; Paillard et al. 2004). Marker gwm639 chromosome 3Sl, further suggesting that different genes on on chromosome 5B was also found associated with the the short arm of chromosome 3Sl confer resistance to O. QTL for Cephalosporium stripe, another important winter yallundae and O. acuformis independently. wheat disease in the PNW (Quincke et al. 2011). Overall, The distal portion of the short arm of Ae. longissima the long arms of homoeologous group 5 contain quantita- chromosome 3Sl was also reported to carry powdery mil- tive resistance to several wheat diseases including eyespot, dew resistance gene Pm13 (Cenci et al. 2003), which is in a possibly representing homoeoloci. similar region as the eyespot resistance found in this study. During QTL mapping of resistance to O. yallundae, a Resistance to both O. yallundae and O. acuformis also was homoeolocus of Pch1 and Pch2, Q.Pch.wsu-7Sl, was iden- found on homoeologous chromosome 3V of Dasypyrum tified at the distal end of chromosome 7SlL (Sheng et al. villosum (Uslu et al. 1998). Our QTL mapping study pro- 2012). Screening Ae. longissima ditelosomic addition and vides further evidence that the short arm of Ae. longissima substitution lines located the resistance to both O. yallun- chromosome 3Sl contains genes for disease resistance, dae and O. acuformis on the long arms of Ae. longissima especially eyespot. chromosome 7Sl (Sheng and Murray 2013). However, there Q.Pch.wsu-5Sl associated with resistance to O. acu- was no evidence of resistance to O. acuformis on chromo- formis was mapped on Ae. longissima chromosome 5Sl some 7Sl in the present study. Pch1 on chromosome 7DL, with close linkage to six SSR markers. Q.Pch.wsu-5Sl a single dominant gene, was effective to both species (Burt associated with resistance to O. yallundae was mapped in et al. 2010). Pch2 on chromosome 7AL is a single partially

1 3 Theor Appl Genet dominant gene (Strausbaugh and Murray 1989) that was References more effective to O. acuformis than to O. yallundae (Burt et al. 2010). Klos et al. (2014) confirmed that Pch2 on Cap- Allan RE, Peterson CJ, Rubenthaler GL, Line RF, Robert DE (1989) pelle Desprez chromosome 7A conferred some resistance Registration of ‘Madsen’ wheat. Crop Sci 29:1575–1576 l Burt C, Hollins TW, Powell N, Nicholson P (2010) Differential against both species. Q.Pch.wsu-7S detected on Ae. lon- seedling resistance to the eyespot pathogens, Oculimacula yal- gissima chromosome 7Sl from our previous study (Sheng lundae and Oculimacula acuformis, conferred by Pch2 in wheat et al. 2012) was only effective to O. yallundae. These and among accessions of Triticum monococcum. Plant Pathol results imply that genes from homoeoloci could react dif- 59:819–828 Burt C, Hollins TW, Nicholson P (2011) Identification of a QTL con- ferently to the two eyespot pathogens. Thus, it is important ferring seedling and adult plant resistance to eyespot on chromo- to screen lines for resistance to each species separately to some 5A of Cappelle Desprez. Theor Appl Genet 122:119–128 provide a gene or genes for both pathogens to the breeding Cenci A, D’Ovidio R, Tanzarella OA, Ceoloni C, Pasquini M, Por- programs. ceddu E (2003) Genetic analysis of the Aegilops longissima 3S chromosome carrying the Pm13 resistance gene. Euphytica Each QTL to O. acuformis detected in this study had 130:177–183 an additive effect. The genotype containing all QTL had Crous PW, Groeneward JZ, Gams W (2003) Eyespot of cereals revis- significantly lower disease severity than all other QTL- ited: ITS phylogeny reveals new species relationships. Eur J Plant containing genotypes except one. All two QTL genotypes Pathol 109:841–850 de la Peña RC, Murray TD (1994) Identifying wheat genotypes had more effective eyespot resistance than one QTL geno- resistant to eyespot disease with a β-glucuronidase-transformed types. Although Q.Pch-oa.wsu-5Sl had a strong effect, the strain of Pseudocercosporella herpotrichoides. Phytopathology combination of Q.Pch-oa.wsu-1Sl and Q.Pch-oa.wsu-3Sl 84:972–977 had greater resistance than other combinations contain- de la Peña RC, Murray TD, Jones SS (1997) Identification of an l RFLP interval containing Pch2 on chromosome 7AL of wheat. ing Q.Pch-oa.wsu-5S by GUS score. Thus, different QTL Genome 40:249–252 combinations demonstrated interactions. Doussinault G, Delibes A, Sanchez-Monge R, Garcia-Olmedo F We found three QTL associated with resistance to O. (1983) Transfer of a dominant gene for resistance to eyespot dis- acuformis and four QTL to O. yallundae from the same Ae. ease from a wild grass to hexaploid wheat. Nature 303:698–700 Friebe B, Tuleen N, Jiang J, Gill BS (1993) Standard karyotype of longissima RIL population. These results were not surpris- Triticum longissimum and its cytogenetic relationship with T. aes- ing because selection of the donor parent (Ae. longissima tivum. Genome 36:731–742 PI 542196) was based on its resistance to both O. yallun- Gervais L, Dedryver F, Morlasis JY, Bodusseau V, Negre S, Bilous dae and O. acuformis. PI 542196 is a useful source of eye- M, Groos C, Trottet M (2003) Mapping of quantitative trait loci for field resistance to Fusarium head blight in an European winter spot resistance and the markers associated with these seven wheat. Theor Appl Genet 106:961–970 QTL should prove useful in marker-assisted introgression Gomez KA, Gomez AA (1984) Statistical procedures for agricultural into adapted wheat lines, addressing the current lack of research. Wiley, New York, pp 319–322 genetic diversity of eyespot resistance. However, the nega- Hollins TW, Lockley KD, Blackman JA, Scott PR, Bingham J (1988) Field performance of Rendezvous, a wheat cultivar with resist- tive effects of linkage drag must be considered and evalu- ance to eyespot (Pseudocercosporella herpotrichoides) derived ated during introgression. from Aegilops ventricosa. Plant Pathol 37:251–260 Jahier J, Doussinault G, Dosba F, Bourgeois F (1979) Monosomic Author contributions HS conducted all the experiments analysis of resistance to eyespot in the variety ‘Roazon’. In: Proceedings 5th international wheat genetics symposium, New of this study, analyzed the data, and wrote the manuscript. Delhi, India, pp 437–440 DRS provided the guidance for the genotyping work and Johnson R (1992) Past, present and future opportunities in breeding for revised the manuscript. TDM supervised the entire project, disease resistance, with examples from wheat. Euphytica 63:3–22 and revised the manuscript several times. Jones SS, Murray TD, Allan RE (1995) Use of alien genes for the development of disease resistance in wheat. Ann Rev Phytopathol 33:429–443 Kimber G (1967) The incorporation of the resistance of Aegilops ven- Acknowledgments PPNS # 0642, Department of Plant Pathology, tricosa to Cercosporella herpotrichoides into Triticum aestivum. College of Agricultural, Human, and Natural Resource Sciences, J Agric Sci 68:373–376 Agricultural Research Center, Hatch Project No. WNP00670, Wash- Klos KLE, Wetzel HC, Murray TD (2014) Resistance to Oculimacula ington State University, Pullman, WA 99164-6430, USA. We thank yallundae and Oculimacula acuformis is conferred by Pch2 in the Washington Grain Alliance for financial support of this research, wheat. Plant Pathol 63:400–404 the USDA National Small Grains Collection for providing seeds of Law CN, Scott PR, Worland AJ, Hollins TW (1976) The inherit- Ae. longissima, and the USDA-ARS Regional Small Grains Genotyp- ance of resistance to eyespot (Cercosporella herpotrichoides) in ing Laboratory at Pullman, WA for assistance with marker analysis. wheat. Genet Res Camb 25:73–79 Lind V (1999) Variation of resistance to Pseudocercosporella her- Conflict of interest none of the authors has the conflict of interest. potrichoides (Fron) Deighton in wheat genotypes carrying the gene Pch-1. Plant Breed 118:281–287 Ethical standards All experiments and genotyping work were carried Maloy OC, Inglis DA (1993) Diseases of Washington crops. Wash. St. out in Pullman, Washington of USA in compliance with the USA laws. Univ. Coop. Ext., Pullman, pp 21–46

1 3 Theor Appl Genet

Muranty H, Jahier J, Tanguy AM, Worland AJ, Law C (2002) Inherit- Sprague R, Fellowes H (1934) Cercosporella foot rot of winter cere- ance of resistance of wheat to eyespot at the adult stage. Plant als. Tech Bull USDA 428:1–24 Breed 121:536–538 Strausbaugh CA, Murray TD (1989) Inheritance of resistance to Murray TD (2010) Eyespot (strawbreaker foot rot). In: Bockus WW, Pseudocercosporella herpotrichoides in three cultivars of winter Bowden RL, Hunger RM, Murray TD, Smiley RW, Morrill W wheat. Phytopathology 79:1048–1053 (eds) Compendium of wheat diseases and insects, Third Edition. Uslu E, Miller TE, Rezanoor NH, Nicholson P (1998) Resistance of APS Press, Minneapolis, pp 32–34 Dasypyrum villosum to the cereal eyespot pathogens, Tapesia Paillard S, Schnurbusch T, Tiwari R, Messmer M, Winzeler M, Keller yallundae and Tapesia acuformis. Euphytica 103:203–209 B, Schachermayr G (2004) QTL analysis of resistance to Fusar- Van Slageren MW (1994) Wild wheats: a monograph of Aegilops L. ium head blight in Swiss winter wheat (Triticum aestivum L.). and Amblyopyrum (Jaub. & Spach) Eig (). Wageningen, Theor Appl Genet 109:323–332 the Netherlands, pp 1–7 Quincke MC, Peterson CJ, Zemetra RS, Hansen JL, Chen J, Riera- Wang SC, Basten J, Zeng ZB (2010) Windows QTL cartographer Lizarazu O, Mundt CC (2011) Quantitative trait loci analysis for 2.5. Department of Statistics, North Carolina State University, resistance to Cephalosporium stripe, a vascular wilt disease of Raleigh, NC. (http://statgen.ncsu.edu/qtlcart/WQTLCart.htm) wheat. Theor Appl Genet 122:1339–1349 Worland AJ, Law CN, Hollins TW, Koebner RD, Giura A (1988) Sheng H, Murray TD (2013) Identifying new sources of resistance to Location of a gene for resistance to eyespot (Pseudocercosporella eyespot of wheat in Aegilops longissima. Plant Dis 97:346–353 herpotrichoides) on chromosome 7D of bread wheat. Plant Breed Sheng H, See DR, Murray TD (2012) Mapping QTL for resistance 101:43–51 to eyespot of wheat in Aegilops longissima. Theor Appl Genet Yildirim A, Jones SS, Murray TD (1998) Mapping a gene confer- 125:355–366 ring resistance to Pseudocercosporella herpotrichoides on chro- Somers DJ, Tsaac P, Edwards K (2004) A high-density microsatel- mosome 4 V of Dasypyrum villosum in a wheat background. lite consensus map for bread wheat (Triticum aestivum L.). Theor Genome 41:1–6 Appl Genet 109:1105–1114 Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the Sprague R (1936) Relative susceptibility of certain species of growth stages of cereals. Weed Res 14:415–421 Gramineae to Cercosporella herpotrichoides. J Agric Res 53:659–670

1 3